FIG. 3.
OPH accumulation and activity are both enhanced significantly by modulating LuxS expression in a coexpression system. (A) OPH was expressed in E. coli W3110 (wild type) and MDAI2 (luxS deficient) by 0.2% arabinose induction and altered AI-2 signaling. That is, MDAI2(pBOL-LacIq) cultures with and without 0.01 mM IPTG were compared with W3110(pBO), MDAI2(pBO), and MDAI2(pBOL) cultures when identical levels of arabinose (0.2%) were added. Throughout, the cell densities (lines) and AI-2 activities (bars) were observed. (B) Transcriptional analysis of luxS for OPH expression in the coexpression system. The RNA was extracted from 1-h.p.i. and 3-h.p.i. samples, and an agarose gel was run to show luxS mRNA levels from RT-PCR using luxS gene-specific primers. (C) After induction, samples were collected and lysed. The OPH activity in each sample was measured and divided by the total protein concentration to derive the specific OPH activity. The data shown here are representative of two independent experiments. The errors shown are standard errors from triplicate OPH activity and total-protein assays. (D and E) OPH accumulation in the soluble (D) and insoluble (E) fractions of cell extracts was examined 1 h.p.i. and 4 h.p.i. by Western blotting. The results shown here are not pooled but, instead, are representative of triplicate experiments (which agreed to within 20%).